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Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours.

Ahlskog JK, Schliemann C, Mårlind J, Qureshi U, Ammar A, Pedley RB, Neri D - Br. J. Cancer (2009)

Bottom Line: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo.In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting.We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, Zurich CH-8093, Switzerland.

ABSTRACT

Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models.

Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology.

Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications.

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Cloning, purification and quality control of anti-CA IX antibodies. (A) The ETH-2-Gold phage antibody library (Silacci et al, 2005) was cloned into pHEN1 vector with VH and VL segments of scFvs flanked by the pelB secretion sequence, and an myc-tag followed by an amber codon and the gene-encoding pIII. (B, E) Schematic representation of the different antibody formats used in this study. ScFv fragments consist of a variable heavy (VH) and a variable light (VL) chain connected by a peptide linker, whereas the small immunoprotein (SIP) (Borsi et al, 2002) is a disulfide-linked homodimer of two scFv–ɛCH4 fusions (C, F) SDS–PAGE analysis of the purified scFv(A3), scFv(CC7) and SIP(A3) and SIP(CC7), respectively, under reducing (R) and non-reducing (NR) conditions. (D) Cloning strategy for the SIP antibodies.
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fig1: Cloning, purification and quality control of anti-CA IX antibodies. (A) The ETH-2-Gold phage antibody library (Silacci et al, 2005) was cloned into pHEN1 vector with VH and VL segments of scFvs flanked by the pelB secretion sequence, and an myc-tag followed by an amber codon and the gene-encoding pIII. (B, E) Schematic representation of the different antibody formats used in this study. ScFv fragments consist of a variable heavy (VH) and a variable light (VL) chain connected by a peptide linker, whereas the small immunoprotein (SIP) (Borsi et al, 2002) is a disulfide-linked homodimer of two scFv–ɛCH4 fusions (C, F) SDS–PAGE analysis of the purified scFv(A3), scFv(CC7) and SIP(A3) and SIP(CC7), respectively, under reducing (R) and non-reducing (NR) conditions. (D) Cloning strategy for the SIP antibodies.

Mentions: ScFvs were converted into the SIP format by cloning VH and VL into pcDNA3.1 (Invitrogen) using the same primers and strategy as described by (Silacci et al, 2006) (Figure 1). The plasmids were transfected into CHO-S cells (Invitrogen) using Cell Line Nucleofector Kit V (Amaxa, Köln, Germany), following the manufacturer's protocol. Transfectomas were grown in RPMI supplemented with 10% FBS and selected by addition of 500 μg ml–1 Geneticin (G418) (Merck Chemicals Ltd, Nottingham, UK). Monoclonal cultures were obtained by fluorescent-activated cell sorting after staining for secreted antibody, as described (Zuberbühler et al, 2008). After 14 days of selection, cells were brought into suspension, and cultured in Power CHO-CD 2 (Lonza, Basel, Switzerland). SIP antibodies were purified from culture medium by affinity chromatography using Protein A Sepharose Fast Flow resin (GE Healthcare), as described by (Zuberbühler et al, 2008).


Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours.

Ahlskog JK, Schliemann C, Mårlind J, Qureshi U, Ammar A, Pedley RB, Neri D - Br. J. Cancer (2009)

Cloning, purification and quality control of anti-CA IX antibodies. (A) The ETH-2-Gold phage antibody library (Silacci et al, 2005) was cloned into pHEN1 vector with VH and VL segments of scFvs flanked by the pelB secretion sequence, and an myc-tag followed by an amber codon and the gene-encoding pIII. (B, E) Schematic representation of the different antibody formats used in this study. ScFv fragments consist of a variable heavy (VH) and a variable light (VL) chain connected by a peptide linker, whereas the small immunoprotein (SIP) (Borsi et al, 2002) is a disulfide-linked homodimer of two scFv–ɛCH4 fusions (C, F) SDS–PAGE analysis of the purified scFv(A3), scFv(CC7) and SIP(A3) and SIP(CC7), respectively, under reducing (R) and non-reducing (NR) conditions. (D) Cloning strategy for the SIP antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736829&req=5

fig1: Cloning, purification and quality control of anti-CA IX antibodies. (A) The ETH-2-Gold phage antibody library (Silacci et al, 2005) was cloned into pHEN1 vector with VH and VL segments of scFvs flanked by the pelB secretion sequence, and an myc-tag followed by an amber codon and the gene-encoding pIII. (B, E) Schematic representation of the different antibody formats used in this study. ScFv fragments consist of a variable heavy (VH) and a variable light (VL) chain connected by a peptide linker, whereas the small immunoprotein (SIP) (Borsi et al, 2002) is a disulfide-linked homodimer of two scFv–ɛCH4 fusions (C, F) SDS–PAGE analysis of the purified scFv(A3), scFv(CC7) and SIP(A3) and SIP(CC7), respectively, under reducing (R) and non-reducing (NR) conditions. (D) Cloning strategy for the SIP antibodies.
Mentions: ScFvs were converted into the SIP format by cloning VH and VL into pcDNA3.1 (Invitrogen) using the same primers and strategy as described by (Silacci et al, 2006) (Figure 1). The plasmids were transfected into CHO-S cells (Invitrogen) using Cell Line Nucleofector Kit V (Amaxa, Köln, Germany), following the manufacturer's protocol. Transfectomas were grown in RPMI supplemented with 10% FBS and selected by addition of 500 μg ml–1 Geneticin (G418) (Merck Chemicals Ltd, Nottingham, UK). Monoclonal cultures were obtained by fluorescent-activated cell sorting after staining for secreted antibody, as described (Zuberbühler et al, 2008). After 14 days of selection, cells were brought into suspension, and cultured in Power CHO-CD 2 (Lonza, Basel, Switzerland). SIP antibodies were purified from culture medium by affinity chromatography using Protein A Sepharose Fast Flow resin (GE Healthcare), as described by (Zuberbühler et al, 2008).

Bottom Line: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo.In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting.We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, Zurich CH-8093, Switzerland.

ABSTRACT

Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models.

Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology.

Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.

Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications.

Show MeSH
Related in: MedlinePlus